The Development and Current Directions of Optogenetics

By Velina Kozareva, THURJ Content

In 1979, Francis Crick identified scientists’ inability to manipulate specific cell types to be a major hurdle in the field of neuroscience; he could only guess that light, through photostimulation, might provide a solution (1). Little did he know that a few decades later, his idea would be realized with the rise of a new field—optogenetics. Before the technology of this area, scientists had two main options for manipulating the function of cells and tissues in the laboratory. One was electrical stimulation, which enabled temporal precision but lacked cell type specificity. The alternative involved genetic modification of cells, which allowed for targeting certain cell types, though without significant temporal control (2).

At the time of Crick’s initial postulation, scientists already knew of light-responsive proteins called opsins, found in both microbes and mammalian eye cells. Microbial opsins encompass a large family of ion pumps and are single-component systems capable of both activation by photons and ion conductance in the cell (2).  They include a variety of bacterial rhodopsins, or opsins bound to retinal, a form of vitamin A. Closely related to these are the channelrhodopsins (ChRs), derived from green algae, and halorhodopsins (NpHRs), derived from halobacteria. Despite this knowledge, it was not until 2005 that a team led by Karl Deisseroth incorporated the single-component channelrhodopsin system into hippocampal neurons, enabling the scientists to manipulate these mammalian cells through light stimulation on a precise time-scale (2). The term “optogenetics” was coined one year later.

The optogenetic “toolbox” of opsins has since been expanded to include many genetically engineered proteins, which were created to boost opsin expression in mammalian cells, increase their range of activation wavelengths, and modify the speed and extent of their activation. This led to the development of step-function opsins in 2009, which display extended activity even after cessation of the light stimulus, by André Berndt’s previous work with Deisseroth at Stanford University (3).

Because these opsins control the excitability of a neuron by changing its membrane potential, optogenetic techniques were initially used to examine neural excitation and inhibition in model organisms. Thereby, neurobiologists could determine the amount of neural excitation necessary for a particular function or behavioral pattern to occur in a specific cell population (3).

Apart from the cellular machinery, the other component of optogenetics is, of course, optics. Encompassing both light delivery and readout, this element of optogenetics presented yet another obstacle for scientists, as they had to determine appropriate light delivery methods for practical and precise control of the targeted cells. Unsurprisingly, current methods differ between in vitro and in vivo studies. For in vitro studies, mercury arc lamps are sometimes used, particularly for examination of brain slices. In vivo experiments require the use of more exact light delivery, in the form of lasers, while mammalian model organisms require the use of optical fibers to direct the light to the desired neurons (2). In addition, more extensive neural networks have been studied in vivo using a combination of photostimulation and functional MRI (3).

From Motor Control to “Smelling” Light

Obviously, the field has come a long way since Crick’s initial speculation. In fact, last year, another major development occurred right here on the Harvard campus. In January 2011, the Samuel Lab of the Center for Brain Science published their work on CoLBeRT, an optogenetic system that allows them to “Control Locomotion and Behavior in Real Time” (4). CoLBeRT uniquely allows neural manipulation in freely moving, unanesthetized organisms like C. elegans. Though the 302 neurons of C. elegans have been extensively mapped, the Samuel Lab wanted to examine patterns of neural activity underlying common behaviors exhibited by these worms.

To target individual motor neurons, they used a digital micromirror device (DMD). Laser lights of the appropriate frequency are reflected off a series of mirrors onto the DMD, which focuses the light with high spatial specificity on the worm (genetically engineered to express ChR2 and NpHR). The laser’s specificity is so precise that the system can illuminate the entire area outside the worm’s body with green light without affecting its motion. As soon as the light is focused on the body, however, neural activity stops, and its muscles relax. The team was also able to stimulate the worms’ egg laying by targeting hermaphrodite-specific motor neurons. The scientists’ fine-tuned control over the worm’s behavior demonstrates the high precision and spatial resolution of the CoLBeRT system.

The lab’s current work focuses on the mechanosensory circuits of C. elegans, which they are investigating through calcium imaging, with a device that Dr. Samuel has termed Dual Mag. This system measures the amount of neural activity in the anterior portion of C. elegans, while simultaneously monitoring the worm’s physical movement. With this device, they can use optogenetic techniques (like CoLBeRT) to determine the specific neurons activated throughout common motor sequences of the worms. Though the lab’s work has focused mainly on studying the neural networks of these simple nematodes, their success suggests that the system can be applied to other simple model organisms or even higher-order neural networks, with some modifications of course.

Yet, as the work of the Murthy Lab of Harvard University demonstrates, optogenetics allows for the study of more than just motor neurons. Their 2012 study in Frontiers in Neural Circuits characterized neural connections of the olfactory system of rodents partly through photostimulation of specific neurons (5). In their project, mice and rats were injected with viruses that induced expression of ChR2. Then, an LED illuminator selectively activated particular olfactory pathways in the brains of the rodents, allowing the researchers to examine interactions between several important regions of the rodents’ olfactory cortex.

Currently, a major question for Dr. Murthy is how the connections within neural circuits allow organisms to distinguish between a variety of sensory stimuli and then act accordingly. To explore this topic, his lab is training mice to differentiate between odors, then determining which parts of the animal’s olfactory cortex are activated during this exercise. Using optogenetic techniques, they can disrupt this learning process and verify which neural pathways are necessary for this complex sensory perception. According to Dr. Murthy, greater knowledge about this process in model organisms, like rodents, could help us better understand the neural circuitry of human sensory perception as well.

Optogenetics for Humans?

Thus far, mice have been the most commonly used, and among the most complex, model organism in optogenetics research. There have been attempts to use optogenetic techniques in primates, such as the rhesus macaque, but targeting specific cell types has not yielded comparable precision in controlling physiological responses (3). Nevertheless, results from microbial opsin delivery have been promising, as viral delivery of ChR2 into a primate brain was successfully performed without eliciting a significant immune response. Furthermore, these results indicate the potential of harnessing these delivery methods to create optogenetics-based therapies for humans.

For people suffering from muscle disease or paralysis, optogenetic techniques could provide an alternative, and perhaps more effective, method of peripheral nerve stimulation, a commonly used experimental treatment. With an optogenetic approach, specific motor neurons could be targeted to prevent excitation of surrounding tissue, which often results in muscle fatigue (3). Stem cells that highly express channelrhodopsins could be photostimulated to induce specific differentiation with greater precision than current chemical or electrical methods, thus reducing unwanted differentiation in surrounding cell types (3). This development could provide great advances in regenerative medicine, particularly as it applies to neurodegenerative disorders. However, the most realistic therapeutic approach is using optogenetic techniques to restore sight in individuals with retinal degeneration. Studies have already shown that inducing ChR2 expression in blind mice can improve vision (3).

Before any of these ideas can become a reality, though, extensive research on the viability of these procedures in humans must be carried out, and, in many ways, this depends upon improvements in delivery techniques for gene therapy. Even with these barriers to clinical applications, Dr. Murthy believes that society will significantly benefit from basic research by developing a better understanding of how neural circuits function.

Despite being a relatively young field, optogenetics is progressing at a break-neck pace. Novel and engineered opsins are being continually discovered and produced, and, as the work of Dr. Samuel and Dr. Murthy attests, optogenetics is well on its way to elucidating many of the mysteries of neuroscience.

References

  1. Yizhar O, Fenno L, Davidson TJ, Mogri M, Deisseroth K. 2011. Optogenetics in Neural Systems. Neuron. 71(1): 9-34.
  2. Fenno L, Yizhar O, Deisseroth K. 2011. The Development and Application of Optogenetics. Ann. Rev. Neurosci. 34: 389–412.
  3. Rein ML, Deussing JM. 2012. The optogenetic (r)evolution. Mol. Genet. Genomics. 287(2): 95–109.
  4. Leifer AM, Fang-Yen C, Gershow M, Alkema MJ, Samuel ADT. 2011. Optogenetic manipulation of neural activity in freely moving Caenorhabditis elegans. Nat. Methods. 8(2): 147-152.
  5. Hagiwara A, Pal SK, Sato TF, Wienisch M, Murthy VN. 2012. Optophysiological analysis of associational circuits in the olfactory cortex. Front. Neural Circuits. 6(18): 1-19.

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